Biochemistry 1987, 26, 6919-6923
6919
High-Resolution Proton and Carbon- 13 NMR of Membranes: Why Sonicate?? Eric Oldfield,* John L. Bowers, and Jeffrey Forbes School of Chemical Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 Received August 7, 1987
We have obtained high-field (1 1.7-T) proton and carbon-13 Fourier transform (FT) nuclear magnetic resonance ( N M R ) spectra of egg lecithin and egg lecithin-cholesterol (1: 1) multibilayers, using “magic-angle” sample spinning (MASS) techniques, and sonicated egg lecithin and egg lecithin-cholesterol (1:l) vesicles, using conventional FT N M R methods. Resolution of the proton and carbon-13 MASS N M R spectra of the pure egg lecithin samples is essentially identical with that of sonicated samples, but spectra of the unsonicated lipid, using MASS, can be obtained very much faster than with the more dilute, sonicated systems. With the 1:1 lecithin-cholesterol systems, proton MASS N M R spectra are virtually identical with conventional FT spectra of sonicated samples, while with 13CN M R , we demonstrate that most I3C nuclei in the cholesterol moiety can be monitored, even though these same nuclei are essentially invisible, i.e., are severely broadened, in the corresponding sonicated systems. In addition, I3C MASS N M R spectra can again be recorded much faster than with sonicated samples, due to concentration effects. Taken together, these results strongly suggest there will seldom be need in the future to resort to ultrasonic disruption of lipid bilayer membranes in order to obtain high-resolution proton or carbon- 13 N M R spectra. ABSTRACT:
o n e of the early goals of N M R studies of membrane structure has been to obtain a well-resolved NMR spectrum, which permits assignment of all sites present, allowing relaxation ( T z ,T I , TI,) experiments to be carried out, in order to study the dynamics of membrane structure. In addition, more recent goals have included obtaining information on the spatial proximity of various groups, via two-dimensional NMR spectroscopy, and on order parameters, primarily by using ?H spin-echo methods on labeled samples. Unfortunately, conventional N M R spectra of intact, unlabeled cell membranes, lipid-water, or lipid-cholesterol dispersions are basically uninformative lumps, which do not permit these types of study. It was shown early on, however, by Chapman et al. (Chapman & Penkett, 1966; Penkett et al., 1968; Chapman et al., 1968b) that ultrasonic dispersal of liquid-crystalline bilayers and intact biological membranes (Chapman et al., 1968a; Jenkinson et al., 1969; Kamat et al., 1970; Glaser et al., 1970) yielded small membrane fragments, vesicles, which did yield fairly high resolution spectra. Whether the structures of sonicated fragments are the same as unsonicated membranes and whether the line broadening or narrowing observed reflects intermolecular interactions as opposed to changes in vesicle size (Finer et al., 1972; Gent & Prestegard, 1974; Bloom et al., 1978) have, however, been topics of lively debate. We thus tried some time ago to develop more nonperturbing methods of investigating lipid and membrane structure and reported 13CN M R spectra of unsonicated lipids (Oldfield & Chapman, 1971; Keough et al., 1973), 2H N M R spectra of unsonicated gel and liquid-crystalline lipids, and their interaction with cholesterol (Oldfield et al., 1971), and ‘H “magic-angle” sample-spinning (MASS) NMR spectra of gel and liquid-crystalline lipid dispersions (Chapman et al., 1972). To date, the 2H N M R method has been quite successful and has seen widespread application to structural studies of gel and liquid-crystalline lipids, to the effects of sterols, peptides, and proteins on lipid structure (Seelig & Seelig, 1978; Kang et al., 1979; Seelig et al., 1981) and to proteins themselves (Smith This work was supported in part by the Biophysics Program of the
US.National Science Foundation (Grant DMB 84-16771). 0006-2960/87/0426-6919$01.50/0
& Oldfield, 1984, and references cited therein). However, selective deuteriation is often a difficult, expensive, and time-consuming process, so we have recently sought to reinvestigate the other nonperturbing techniques, I3C and ‘H NMR, using state-of-the-art solid-state NMR instrumentation. In particular, we have followed up on the 1972 prediction that 13C MASS NMR at high fields should be particularly informative (Chapman et al., 1972). In this paper we report the results of our recent ‘H and 13C MASS N M R studies of two archetypal model membrane systems: egg lecithin and egg lecithin-cholesterol (l:l), hand dispersed in excess water. The results indicate that resolution is at least as good as with sonicated samples, but because dilute samples are not required, spectra may be obtained in much shorter time periods, typically at least an order of magnitude faster, with MASS. In addition, our results demonstrate that numerous resonances in the 13CNMR spectrum of cholesterol (in egg lecithin), which are “invisible” in sonicated dispersions, give very narrow lines in 13C MASS experiments, so that ultrasonic dispersal of lipids (or other membranes) now seems to be an unnecessary step in obtaining highly resolved ‘H and 13C NMR spectra of membranes.
EXPERIMENTAL PROCEDURES N M R Spectroscopy. All ‘H and I3C NMR spectra were obtained on a “home-built” NMR spectrometer, which operates at 500 MHz for ‘H, using an Oxford Instruments (Osney Mead, U.K.) 2.0411. bore, 11.7-T superconducting solenoid, together with a Nicolet (Madison, WI) Model 1280 computer system, a Henry Radio (Los Angeles, CA) Model 1002 radio frequency amplifier, an Amplifier Research (Souderton, PA) Model 200L radiofrequency amplifier, and either a Doty Scientific (Columbia, SC) MASS NMR probe or a 5-mm multinuclear solution N M R probe (Cryomagnet Systems, Indianapolis, IN). For ‘H MASS experiments, the 90’ pulse widths were 8 p s , and spinning rates were -2.4-2.8 kHz. For sonicated samples the ‘H 90’ pulse width was 12 w.For 13CMASS, dipolar decoupled (40-W ‘H power) Bloch decays were recorded using 9-ps 13C 90° pulse widths. For 13CNMR of sonicated samples, 10-ps pulse widths were used, 0 1987 American Chemical Society
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together with Waltz-16 proton decoupling (gated at 0.6 and 5 W), to ensure full decoupling and nuclear Overhauser effect, but without appreciable sample heating. Temperature was adjusted via a gas-flow cryostat, and values reported were gas-flow temperatures monitored with a Doric (San Diego, CA) Trendicator. All spectra were referenced to an external standard of tetramethylsilane, and high-frequency, low-field, deshielded, paramagnetic shifts are denoted as positive (IUPAC 6 scale). Lipid Samples. Egg yolk lecithin (EYL) was obtained from Lipid Products (South Nuttfield, Surrey, U.K.) and was used without further purification. Cholesterol (CHOL) was from Aldrich (Milwaukee, WI) and had been recrystallized 3 times from EtOH before use. EYL samples were prepared by addition of the appropriate amount of "100%" D 2 0 (Sigma, St. Louis, MO) to dried EYL and either dispersed by hand or for three 6-min periods using a Heat Systems-Ultrasonics ultrasonicator (Plainview, NY). Sample temperature was kept low by having the sonicating vial surrounded by ice, and by agitating the sample for 2 min in the ice, between sonic bursts. EYL-CHOL samples were prepared by codissolving the appropriate amounts of EYL and CHOL in CHCl, and removing the solvent under an N2 stream at -40 OC, followed by evacuation over P4Ol0for 24 h. The samples were then hydrated to the appropriate level (50 wt % lipid for an unsonicated dispersion, 10 wt % for a sonicated dispersion) and dispersed, basically as described above for EYL. RESULTSAND DISCUSSION We show in Figure 1 the 500-MHz IH N M R spectra of EYL and EYL-CHOL (1:l) samples, in excess water, using 'H MASS (Figure 1A,C) or conventional high-resolution N M R of sonicated samples (Figure lB,D). As can be seen by comparison of parts A and B of Figure 1, the 'H MASS N M R spectrum of a 50 wt % hand dispersion (multilayers, Figure 1A) is virtually identical with that obtained with a dilute, sonicated dispersion (Figure 1B). We discuss the origin of the high-resolution MASS N M R spectrum, which is accompanied by several sharp spinning side bands (not shown), in detail elsewhere (Forbes et al., 1987). For completeness we comment on it briefly again, since the observation of a high-resolution 'H MASS NMR spectrum is rather surprising, because the static dipolar Hamiltonian EDij(4)(3Z2iZ2j- Ii'Ij) i
